Identification and Functional Analysis of a Delta Class Glutathione S

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Agricultural and Environmental Chemistry

Identification and Functional Analysis of a Delta Class Glutathione S-transferase Gene Associated with Insecticide Detoxification in Bradysia odoriphaga Bowen Tang, Wu Dai, Lijun Qi, Qi Zhang, and Chunni Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b02874 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 15, 2019

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Journal of Agricultural and Food Chemistry

Identification and Functional Analysis of a Delta Class Glutathione S-transferase Gene Associated with Insecticide Detoxification in Bradysia odoriphaga Bowen Tang, Wu Dai, Lijun Qi, Qi Zhang, Chunni Zhang* State Key Laboratory of Crop Stress Biology for Arid Areas, and Key Laboratory of Integrated Pest Management on Crops in Northwestern Loess Plateau, Ministry of Agriculture, College of Plant Protection, Northwest A&F University, Yangling 712100, Shaanxi, China

*Corresponding author. Chunni Zhang [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

2

A delta class GST gene (BoGSTd2) is identified from Bradysia odoriphaga for

3

the first time. Developmental expression analysis showed expression of BoGSTd2 is

4

significantly higher in the fourth instar larval stage and in the adult stage.

5

Tissue-specific expression analysis found that BoGSTd2 was expressed predominantly

6

in the midgut and Malpighian tubules in the fourth-instar larvae and in the abdomen

7

of adults. Expression of BoGSTd2 was significantly upregulated following exposure

8

to chlorpyrifos and clothianidin. In vitro inhibition and metabolic assays indicated that

9

recombinant BoGSTd2 could not directly metabolize chlorpyrifos and clothianidin.

10

Nevertheless, disk diffusion assays indicated that BoGSTd2 plays an important role in

11

protection against oxidative stress. RNAi assays showed that BoGSTd2 participates in

12

the elimination of ROS induced by chlorpyrifos and clothianidin. These results

13

strongly suggest that BoGSTd2 plays an important role in chlorpyrifos and

14

clothianidin detoxification in B. odoriphaga by protecting tissues from oxidative

15

stress induced by these insecticides.

16 17

KEYWORDS:

18

detoxification; RNA interference; peroxidase activity

Bradysia

odoriphaga;

glutathione

2


S-transferases;

insecticide

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19

INTRODUCTION

20

The Chinese chive maggot, Bradysia odoriphaga Yang et Zhang (Diptera:

21

Sciaridae) is a serious crop pest, feeding on more than 30 plant species from seven

22

families, especially Chinese chives (Allium tuberosum).1,2 Larvae of B. odoriphaga

23

cluster in the roots and stems, causing severe damage.3,4,5 The application of

24

insecticides is the main method for control of this pest.3,6 However, the extensive

25

application of chemical insecticides has often led to development of insecticide

26

resistance in field populations of B. odoriphaga. Studies showed that all eight of the

27

tested populations developed moderate to high resistance to phoxim and

28

chlorpyrifos, and five out of eight populations displayed a moderate resistance to

29

clothianidin.6 In general, insecticide resistance in insects has been associated with

30

increased activity of detoxification enzymes including glutathione S-transferases

31

(GSTs), cytochrome P450 monooxygenases (P450) and carboxylesterase (CarE).7-9

32

Glutathione S-transferases (GSTs) are a major family of phase II detoxification

33

enzymes found in almost all living organisms.10-11 They can catalyze the conjugation

34

of electrophilic endogenous and exogenous compounds with the thiol group of

35

reduced glutathione (GSH), increasing the solubility of the resultant products and thus

36

facilitating excretion from the cell.12,13 GSTs are highly diversified and play important

37

roles in the detoxification of a wide range of xenobiotic and endogenous compounds,

38

and also involves in protection against oxidative stress.12,13 The delta and epsilon

39

classes are insect-specific14 and play important roles in metabolizing insecticides and

40

conferring resistance to many classes of insecticides in insects.9,13,15 In Drosophila 3



41

melanogaster, DmGSTd1 is capable of metabolizing the insecticide DDT.16 A recent

42

study showed that BdGSTd1 and BdGSTd10 play significant roles in the

43

detoxification of malathion in Bactrocera dorsalis.17 CpGSTd1 and CpGSTd3 in

44

Cydia pomonella were involved in metabolism of lambda-cyhalothrin.18,19 A number

45

of GSTs display peroxidase activity that protects tissues or cells against oxidative

46

damage and oxidative stress.9,20,21 BgGSTD1 from Blattella germanica exhibited the

47

highest peroxidase activity in protecting against oxidative stress.22 PcGSTd1 from

48

Panonychus citri plays an antioxidant role by reducing oxidative stress caused by

49

fenpropathrin.23 Previous studies demonstrated that a delta class GST from

50

Nilaparvata lugens contributed to the resistance by detoxifying lipid peroxidation

51

products caused by pyrethroids.24 However, information on GST genes in B.

52

odoriphaga is rather limited and the properties of specific GST genes and related

53

insecticide detoxification mechanisms in this insect are scarce.

54

To elucidate whether delta class GSTs contribute to insecticide detoxification in

55

B. odoriphaga, a delta class GST gene (BoGSTd2) was cloned and identified from B.

56

odoriphaga. The spatio-temporal expression profiles of BoGSTd2 were analyzed by

57

quantitative real time PCR (qRT-PCR). The expression of BoGSTd2 was also studied

58

after treatment with insecticides. We functionally expressed the BoGSTd2 in

59

Escherichia coli and examined biochemical properties of the purified recombinant

60

protein, performed in vitro inhibition and metabolic assay of recombinant BoGSTd2

61

to elucidate its capability to metabolize insecticides, and further investigated the

62

potential roles of recombinant BoGSTd2 in antioxidant defense. RNAi mediated by 4


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63

double-stranded RNA (dsRNA) was used to study gene function. Additionally,

64

biochemical assay was performed to infer a link between BoGSTd2 and the oxidative

65

effect induced by insecticides. Our results reveal new insights into the molecular

66

mechanism of delta class GST response to insecticide detoxification in B. odoriphaga.

67

MATERIALS AND METHODS

68

Insect Rearing

69

A laboratory population of B. odoriphaga was reared for more than 70

70

generations on fresh chive stems without exposure to any insecticides and maintained

71

at 25±1 °C, 70±5% relative humidity with a photoperiod of 16:8 h (L : D).

72

Sample Collection

73

For study of gene expression patterns, 30 individuals for 1st-instar larvae, 20

74

individuals for 2nd to 4th-instar larvae, 20 pupae and 20 adults were collected.

75

Various tissues from 150 adults (thorax, abdomen, head, legs, wings and antennae) as

76

well as from 150 4th-instar larvae (head, midgut, malpighian tubule, fat body and the

77

rest tissue) were dissected under a microscope. All samples were immediately

78

immersed in liquid nitrogen.

79

Insecticide Treatments

80

Lambda-cyhalothrin

(Analytical

standard,

CAS

number:

91465-08-6),

81

chlorpyrifos (99%, CAS number: 2921-88-2), clothianidin (Analytical standard, CAS

82

number: 210880-92-5) and Triton X-100 (CAS number: 9002-93-1) were purchased

83

from Aladdin (China Shanghai). Acetone (>99.5%) was purchased from Kermel

84

(Tianjin, China). Each insecticide was dissolved in acetone and diluted to the LC30 5



85

concentrations (8.21 mg/L for Lambda-cyhalothrin, 2.21 mg/L for chlorpyrifos, and

86

0.90 mg/L for clothianidin)25 with distilled water containing 0.05% (v/v) Triton

87

X-100 for bioassays. Bioassays were conducted on 3rd-instar larvae of B. odoriphaga

88

using a standard contact and stomach bioassay method.6 Fresh Chinese chive stems

89

were cut into pieces and dipped into insecticide solutions for 30 s, and then air-dried

90

in the shade. The 3rd-instar larvae of B. odoriphaga were dipped into an insecticide

91

solution for 10 s and then transferred to a 50 mm Petri dish with filter paper. Three

92

replicates were conducted with at least 20 larvae per treatment. The insects were

93

reared in uniform environments as described above. Distilled water containing 0.05%

94

(v/v) Triton X-100 was used as the control. The surviving larvae were collected into

95

RNase-free tubes at 12, 24, 36 or 48 h post-treatment and immediately immersed in

96

liquid nitrogen.

97

Total RNA Isolation and cDNA Synthesis

98

Total RNA was extracted using the RNAiso plus Reagent (TaKaRa, Japan)

99

according to the manufacturer's instructions. RNA quality was determined by 1%

100

agarose gel electrophoresis and then quantified using a Nanodrop™ 1000

101

(Thermoscientific, Lithuania). First-strand cDNA was synthesized from1μg of total

102

RNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser, Perfect Real Time

103

(TaKaRa, Japan) and then stored at -20 °C.

104

Molecular Cloning of BoGSTd2

105

Primers for conservative fragment amplification of BoGSTd2 were designed

106

based on the conserved nucleotide and amino acid sequences of the gene reported for 6


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107

other insect species and listed in Table S1. Polymerase chain reactions (PCR) were

108

performed using a S-1000 Thermal Cycler (Bio-Rad, Hercules, CA, USA) under the

109

following program: 94 °C for 3 min; 35 cycles of 30 s at 94 °C, 30 s at 52 °C and 60 s

110

at 72 °C and a final extension at 72 °C for 10 min. The amplified PCR products were

111

separated in 2% agarose gel and purified by a Biospin Gel Extraction Kit (Bioer

112

Technology, Hangzhou, China). Purified PCR products were cloned into pMD18-T

113

vector (TaKaRa, Japan) and then sequenced completely in both directions (AuGCT,

114

Beijing, China). Rapid amplification of cDNA ends (RACE) was performed to obtain

115

the full-length cDNA. The first strand cDNA for 5′-and 3′-RACE was synthesized

116

from 1μg of total RNA isolated from 15 third instar larvae using a SMARTer™

117

RACE cDNA Amplification Kit (TaKaRa, Japan). 5′-RACE was amplified by nested

118

PCR using 5-GSP1 and outer Primer for the primary PCR reaction at annealing

119

temperature 56 °C. The second PCR reaction was performed using 5-GSP2 combined

120

with inner Primer at annealing temperature 56 °C. The primary PCR reaction for

121

3′-RACE was performed using 3-GSP1 and Long Primer at annealing temperature

122

56 °C and the second PCR reaction was performed using 3-GSP2 and Short Primer at

123

annealing temperature 58 °C. All PCR cycling parameters were 94 °C for 3 min; 35

124

cycles of 94 °C for 30 s, 30 s at annealing temperature and 72 °C for 60 s; 72 °C for

125

10 min. Based on the sequences obtained from the 3′- and 5′-RACE, the putative

126

full-length of GST gene was amplified with specific primers GST2-F and GST2-R.

127

The PCR products were cloned into the pMD18-T vector (TaKaRa, Japan) and

128

sequenced completely. All primers used are listed in Table S1. 7



129

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Sequence Analysis

130

The deduced protein isoelectric points and molecular mass were predicted by

131

ExPASy Proteomics Server (https://web.expasy.org/compute_pi/). The signal peptide

132

was predicated by SignalP 3.0 Serve (http://www.cbs.dtu.dk/services/SignalP-3.0/).

133

The

134

(http://www.compbio.dundee.ac.uk/jpred/). The G-site and H-site was predicated by

135

InterPro (http://www.ebi.ac.uk/interpro/). Alignment of amino acid sequences was

136

made using DNAMAN software (LynnonBiosoft, USA). The phylogenetic tree was

137

constructed by neighbor-joining method with 1000 bootstrap replicates using MEGA

138

6.0 software according to the amino acid sequences (https://www.megasoftware.net/).

139

The program SWISS-MODEL (https://swissmodel.expasy.org/) was used to predict

140

three-dimensional structure.

141

Quantitative Real-Time PCR

secondary

structure

was

predicated

by

Jpred4

142

qRT-PCR was carried out using a Cycler IQ Real-Time PCR detection system

143

(Bio-Rad, Hercules, CA, USA) with 20 μL volume: 10 μL 2× UltraSYBR Mixture

144

(KWBIO, Beijing, China), 1 μL of each primer (10 mM), 1 μL of template, and 7 μL

145

ddH2O. Amplification program as follows: 95 °C for 10 min; followed by 40 cycles of

146

95 °C for 15 s, and 60 °C for 60 s.

147

was used as a negative control. A melting curve analysis from 55 to 95 °C with

148

increments of 0.5 °C every 30 s was conducted to ensure specificity and consistency

149

of the amplified product. The amplification efficiency (E) was calculated using the

150

equation: E = (10[-1/slope]-1) ×100. Each experiment was performed using three

RNase-free water instead of cDNA templates

8


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151

biological replicates with three technical replications. Data were analyzed by the

152

2−ΔΔCT method26 and normalized using the following combinations of reference genes:

153

18S, Tubulin, and RPS7 for various tissues of adults; 18S and RPS15 for various

154

tissues of larvae; 18S, RPS15 and RPS7 for different developmental stages; and

155

RPL18 and RPS15 for insecticide treatments. Statistical significance of the gene

156

expression data was determined using SPSS 20.0 (IBM Inc., Chicago, Illinois, USA).

157

Prokaryotic Expression and Purification of Recombinant BoGSTd2 Protein

158

The open reading frame (ORF) of BoGSTd2 was sub-cloned into the expression

159

vector pET-28a (+) (Novagen, Madison, WI) between EcoR I and Xho I restriction

160

sites. The resultant plasmid was transformed into E. coli BL21 (DE3) cells. The

161

expression of recombinant proteins was induced with 0.4 mM isopropyl

162

β-d-1-thiogalactopyranoside (IPTG) for 6 h at 37 °C. The cells were harvested and

163

sonicated in ice, then centrifuged at 12,000 g at 4 °C for 20 min. The supernatant was

164

purified using Ni-NTA Resin (TransGen Biotech, Beijing, China). The recombinant

165

GST protein was eluted using a graded series of 20 to 250 mM imidazole dissolved in

166

balance buffer and evaluated by sodium dodecyl sulfate polyacrylamide gel

167

electrophoresis (SDS-PAGE). Purified recombinant protein was dialyzed with 10 mM

168

phosphate buffered saline (PBS) (pH 7.4) overnight on ice. The protein concentrations

169

were determined using a BCA Protein Assay Kit (Heart Biological Technology,

170

Xi’an, China).

171

Enzyme Activity Assays of the Recombinant BoGSTd2

9



172

The

activity

of

recombinant

BoGSTd2

were

Page 10 of 46

determined

using

173

1-Chloro-2,4-Dinitrobenzene (CDNB) as substrate.27 All reactions were performed in

174

100 mM phosphate buffered saline in the presence of 1 mM CDNB, 1 μg of

175

recombinant BoGSTd2

176

96-well microplates (Thermo, USA) with total reaction volume of 200 μL.

177

Absorbance at 340 nm was measured at 5 min intervals using a M200 PRO

178

Microplate Reader (Tecan, Switzerland). The optimum pH for BoGSTd2 activity was

179

investigated at 30 °C, with a pH range of 4.0–8.5. The thermostability of BoGSTd2

180

was assayed by preincubation of the enzyme solution in 100 mM PBS pH 7.0 at

181

varied temperatures for 30 min, then conjugating activity was measured.

and 1 mM glutathione (Sigma-Aldrich, UK) in Nunc

182

The kinetic parameters of recombinant BoGSTd2 were determined by various

183

concentrations (0.05 to 1.6 mM) of CDNB substrate, 1 μg of recombinant BoGSTd2

184

and 1 mM of the GSH constant at the optimal pH and temperature. The reaction

185

without GST protein was designed as a control. Each test was conducted in four

186

replicates. The values of Km and Vmax were determined according to the double

187

reciprocal Lineweaver-Burk plot method using GraphPad Prism 6.0 (GraphPad

188

Software, USA).

189

Glutathione peroxidase (GPOX) activity of BoGSTd2 against cumene

190

hydroperoxide (CHP) or H2O2 (Aladdin, China), two substrate of GSH-dependent

191

peroxidase, was determined according to published method.28 The reactions were

192

performed in 100 mM phosphate buffered saline in the presence of 1mM EDTA, 1

193

mM glutathione, 0.2 mM NADPH, 1 U glutathione reductase, 4 μg BoGSTd2 protein, 10


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194

and various concentrations (0.05 to 1.6 mM) of CHP or H2O2. The reaction mixture

195

was preincubated at 25°C for 5 min prior to the addition of CHP or H2O2. A negative

196

control was carried out using PBS instead of BoGSTd2 protein. Absorbance at 340

197

nm was measured using a M200 PRO Microplate Reader (Tecan, Switzerland). Each

198

test was conducted in at least three replicates. The Michaelis constant (Km) and Vmax

199

were determined with Lineweaver–Burk plots method using GraphPad Prism 6.0

200

(GraphPad Software, USA).

201

The half-inhibitory concentration (IC50) of S-hexylglutathione (GTX)

202

(Sigma-Aldrich), a known GSTs inhibitor,19 was measured using different

203

concentrations of GTX (0, 0.016, 0.08, 0.4, 2, 10 and 50 μM) (Dissolved in acetone).

204

1 μg of recombinant BoGSTd2 protein and different concentrations of GTX were

205

pre-incubated in 100μL of 100 mM PBS at 30 °C for 5 min. The mixture was then

206

added to the reaction of 1 mM GSH and 0.1 mM CDNB in a total volume of 200 μl of

207

100 mM PBS buffer (pH 7.0). For the inhibition assay of insecticides against

208

BoGSTd2, lambda-cyhalothrin, clothianidin and chlorpyrifos were used in a range of

209

0.32–1000 μM. All reactions were monitored by measuring absorbance as described

210

above. Each test was conducted in four replicates and samples with 1μg of denatured

211

BoGSTd2 were used as negative controls. The IC50 values were determined using

212

Graphpad Prism 6.0 (GraphPad Software, USA).

213

Metabolism Assays

214

The insecticide metabolism assay of recombinant BoGSTd2 was performed

215

according to the method described by Kostaropoulos et al.29 The UPLC assay was 11



216

conducted on Shimadzu LC-30A (SHIMADZU, Japan). The reaction system

217

containing 0.1 M PBS (pH 7.4), 20 μg recombinant protein, 2.5 mM GSH and 200

218

mg/L insecticide in a total volume of 1 mL was incubated at 30 °C with shaking at

219

200 rpm for 120 min, 1 mL of methanol (HPLC grade) was added to stop the reaction.

220

The reaction mixture was then centrifuged at 12 000 g for 10 min at 4 °C and 10 μL

221

supernatant was absorbed into a InertSustain AQ-C18 (2.1×100 mm, 3 μm)

222

(SHIMADZU, Japan). Lambda-cyhalothrin was separated by mobile phase of 80%

223

methanol and 20% water with a 0.8 mL/min of flow rate and a absorbance wavelength

224

of 220 nm at 30 °C. Chlorpyrifos were separated by mobile phase of 85% methanol

225

and 15% water with a 0.8 mL/min of flow rate and a absorbance wavelength of 290

226

nm at 20 °C. Clothianidin was separated by mobile phase of 37.5% methanol and

227

62.5% water at 0.6 mL/min of flow rate with a absorbance wavelength of 265 nm at

228

35 °C. The heat-inactivated GST was used as control. The experiments were

229

performed in triplicate.

230

Disk Diffusion Assay

231

A disk diffusion assay was carried out following Yan et al.30 200 μL of the E.

232

coli BL21 (DE3) culture (OD600 = 0.8) containing overexpressed BoGSTd2 was

233

plated onto LB agar plates and incubated at 37 °C for 1 h. Filter discs with 6-mm

234

diameter soaked with various concentrations of cumene hydroperoxide (0, 25, 50, 100,

235

and 200 mM) were placed on the agar plates and incubated at 37 °C for 24 h. E. coli

236

BL21 (DE3) with the pET-28a (+) (no insert) was used as the control and treated

237

under the same conditions. The inhibition zones were measured. The test was 12


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238

conducted in three replicates and data were analyzed using SPSS 20.0 (IBM Inc.,

239

Chicago, Illinois, USA).

240

Double-stranded RNA (dsRNA) Synthesis

241

BoGSTd2 dsRNA fragment was synthesized using the T7 RiboMAXTM

242

Express RNAi System (Promega) followed the manufacturer’s instructions. The

243

fragments were amplified using primers contained a T7 RNA polymerase promoter

244

(Table S1). The purified PCR fragments were used as template for dsRNA synthesis.

245

Synthesized dsRNA was purified according to the manufacturer’s instructions and

246

stored at -80 °C until use. Double strand green fluorescent protein (dsGFP) was used

247

as a control.

248

RNA Interference and Bioassays

249

For RNAi assay, third-instar larvae were fed with artificial diet containing 30

250

μg/g dsGSTd2 or dsGFP (control). After 48 h, 10 individuals were collected and

251

immediately immersed in liquid nitrogen. The silence efficiency of BoGSTd2 was

252

measured by qRT-PCR as described above.

253

In order to evaluate sensitivity of dsGSTd2-treated insects to insecticide,

254

lambda-cyhalothrin, chlorpyrifos and clothianidin were used in the bioassays.

255

Distilled water containing 0.05% (v/v) Triton X-100 was used as the negative control.

256

B. odoriphaga third-instar larvae were initially fed on an artificial diet containing

257

dsGSTd2 or dsGFP for 48 h, and then fed on an artificial diet containing insecticide at

258

LC30 dosage. Mortality of B. odoriphaga larvae was assessed at 48 h post-exposure to

259

the insecticides. Each treatment contained five replicates (at least 20 larvae for per 13



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260

replicate). Statistical analyses were determined by t-test in SPSS 20.0 (IBM Inc.,

261

Chicago, Illinois, USA).

262

Measurement of Reactive Oxidative Species in B. odoriphaga

263

The generation of reactive oxidative species (ROS) in the larvae exposed to

264

insecticides

265

(DCFH-DA) according to the method of Maharajan et al.31 10 larvae were washed and

266

homogenized in 300 μL ice cold 10 mM PBS (pH 7.4). The homogenate was

267

centrifuged at 12 000 g for 20 min at 4 °C, and then 20 μL supernatant was added to a

268

Flat Black 96-well plate (Thermo, USA) and incubated at room temperature for 5

269

min. Subsequently, 100 μL PBS containing 10 μM DCFH-DA were added and

270

incubated in the dark at 37 °C for 30 min. The fluorescence intensity was determined

271

with excitation at 488 nm and emission at 525 nm using a M200 PRO Microplate

272

Reader (Tecan, Switzerland). Each test was conducted in three replicates and PBS

273

was used as negative control. The ROS level was expressed as relative fluorescence

274

intensity.

275

Measurement of Glutathione Peroxidase Activity in B. odoriphaga

were

measured

using

2,7-Dichlorodi-hydrofluorescein

diacetate

276

GPOX activity following insecticide treatment was measured as described

277

above using 20 μL insect homogenate. 10 larvae were washed and homogenized in

278

300 μL ice cold 0.1 M PBS (pH 7.4). All reactions were performed using 1 mM CHP

279

as substrate. Protein content was measured as described above. The results are

280

expressed in μmol of NADPH mg of protein−1 min−1.

281

RESULTS 14


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282


cDNA Sequence Analysis of BoGSTd2

283

The full length cDNA sequence of BoGSTd2 was obtained from B. odoriphaga

284

and submitted to GenBank (GenBank accession numbers: KX254568; Figure S1).

285

The full length cDNA sequence of BoGSTd2 is 916 bp and contains a 642 bp open

286

reading frame (ORF), which encodes a protein of 213 amino acids with a predicted

287

molecular mass of 23.78 kDa and a theoretical pI of 5.12. The amino acid sequence of

288

BoGSTd2 shares a high identity with the GST delta subfamily known from other

289

insects (Figure 1). The full-length BoGSTd2 protein showed the highest similarity to

290

AdGSTd1 (Anopheles darlingi, ETN67358.1, 55.61% identity), followed by

291

AgGSTd1-5 (Anopheles gambiae, CAB03592.1, 55.14% identity), CpGSTd (Culex

292

pipiens,

293

ABG56084.1, 54.46% identity), DmGSTd1 (Drosophila melanogaster, AAB26519.1,

294

53.99% identity) and DaGSTd1 (Delia antiqua, ALF04571.1, 53.70% identity). The

295

3D structure of BoGSTd2 shows that it consists of 9 α-helices and 5 β-sheets (Figure

296

S2). BoGSTd2 includes an N-terminal domain (residues 3–77, with βαβαββα

297

topology) which contains a key serine residue acid (residue 11) and a C-terminal

298

domain (residues 91–209). Two active domains, the GSH-binding site (G-site) and a

299

putative substrate-binding site (H-site), are located in the N-terminal and C-terminal,

300

respectively (Figure S1).

AEW07373.1,

54.63%

identity),

MdGSTd

(Mayetiola

destructor,

301

The phylogenetic tree was constructed on the basis of amino acid sequences of

302

known insect GST genes. The results grouped various GSTs into seven large clusters

303

(delta, epsilon, omega, sigma, theta, zeta, and unclassified classes). BoGSTd2 was 15



304

clustered in the delta class of GSTs, most closely related to the dipterous delta class

305

GSTs subcluster (Figure 2).

306

Developmental-dependent and Tissue-specific Expression of BoGSTd2

307

The expression pattern of BoGSTd2 in different developmental stages was

308

determined by qRT-PCR. The results indicated that BoGSTd2 is expressed at all

309

tested developmental stages of B. odoriphaga. The expression of BoGSTd2 was

310

significantly higher in the fourth instar larva and adult stage (Figure 3A). Compared

311

to eggs, the expression of BoGSTd2 was 5.54-fold in the fourth instar larva stage,

312

6.03-fold in the male adult and 4.02-fold in the female adult, respectively.

313

Transcript levels of the BoGSTd2 were measured in various tissues from adults

314

(antennae, head, thorax, abdomen, legs and wings) as well as from 4th-instar larvae

315

(head, midgut, malpighian tubule, fat body and the rest tissue). The results revealed

316

that BoGSTd2 was expressed predominantly in the abdomen of adults (Figure 3B).

317

Tissue-specific expression analysis showed that BoGSTd2 was expressed differently

318

in various tissues of the fourth-instar larvae (Figure 3C). BoGSTd2 was expressed

319

primarily in the midgut, followed by Malpighian tubules, and much lower in the head,

320

fat body, and the remaining tissues (Figure 3C).

321

Effect of Insecticide Exposure on Expression of BoGSTd2

322

The expression levels of BoGSTd2 after exposure to lambda-cyhalothrin,

323

chlorpyrifos and clothianidin at concentrations of LC30 were measured by qRT-PCR

324

(Figure 4). Expression is significantly lower than that of the control at 24 h

325

post-exposure to LC30 of lambda-cyhalothrin, but no significant differences were 16


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326

observed at 12 h, 36 h and 48 h post-treatment compared with the control (Figure

327

4A). Compared with the control, BoGSTd2 expression was significantly upregulated

328

at 24 h and 48 h post-exposure to LC30 of chlorpyrifos. The mRNA level of BoGSTd2

329

was significantly lower than that of the control at 12 h and 36 h post-exposure to the

330

LC30 chlorpyrifos (Figure 4B). The mRNA level of BoGSTd2 was significantly

331

increased at 24 h and 36 h post-exposure to LC30 clothianidin compared to control

332

(Figure 4C).

333

Bacterial Expression and Purification of BoGSTd2

334

The gene encoding BoGSTd2 was amplified and ligated into the expression

335

vector pET-28a (+). The construct was induced at 37 °C for 6 h with 0.4 mM IPTG in

336

E. coli. SDS-PAGE analysis indicated that recombinant BoGSTd2 was expressed

337

mostly in a soluble form. After purification, the BoGSTd2 was obtained in good yield

338

(approximately 21.25 mg/mL culture). The molecular mass of recombinant BoGSTd2

339

(containing His-Tag) was approximately 25 kDa (Figure S3).

340

Enzymatic Properties of Recombinant BoGSTd2

341

The activity of recombinant BoGSTd2 was measured using the substrate CDNB

342

for GSTs. The recombinant BoGSTd2 at 7.0 of pH exhibited optimum catalytic

343

activity toward CDNB (Figure S4). After recombinant BoGSTd2 was incubated at

344

various temperature ranges from 10 to 60 °C for 30 min, the maximum activity was

345

detected at 30 °C (Figure S4). The kinetic constants for recombinant BoGSTd2 were

346

determined using 1 μg of recombinant BoGSTd2 with GSH held constant at 1 mM

347

and different concentrations (0.05 to 1.6 mM) of CDNB. The results show that the 17



348

Vmax and Km of BoGSTd2 are 349.40±10.71 μmol/min/mg and 0.122±0.012 mM,

349

respectively (Table 1, Figure S5). Inhibition assays were conducted with GST

350

inhibitor GTX in vitro using CDNB as substrate. The IC50 for GTX was 3.769±0.598

351

μM (Figure S4). These results indicate that BoGSTd2 is catalytically active,

352

displaying glutathione transferase activity toward CDNB.

353

Glutathione peroxidase activity of BoGSTd2 (Aladdin, China) was determined

354

using cumene hydroperoxide or H2O2 as substrates (Table 1, Figure S5). The results

355

showed that the Vmax and Km of BoGSTd2 towards CHP were 50.20±3.105

356

μmol/min/mg and 0.367±0.063 mM, respectively. The Vmax and Km of BoGSTd2

357

towards H2O2 were 155.90±9.329 μmol/min/mg and 0.415±0.064 mM, respectively.

358

Inhibition assays were performed to explore the possible interaction of

359

BoGSTd2 with insecticides (lambda-cyhalothrin, chlorpyrifos and clothianidin). The

360

results showed that none of the insecticides significantly inhibited BoGSTd2 activity

361

under assay conditions (Figure S6), indicating that the three insecticides was unable to

362

strongly conjugate with BoGSTd2. UPLC assay showed that the quantities of the

363

three insecticides did not change significantly compared with the control after

364

incubation with recombinant BoGSTd2 (Figure 5 and S7), suggesting that BoGSTd2

365

cannot directly metabolize chlorpyrifos, clothianidin and lambda-cyhalothrin.

366

Disk Diffusion Assay

367

A disk diffusion assay was performed to detect the antioxidant activity of

368

BoGSTd2. E. coli cells overexpressing BoGSTd2 were exposed to CHP which is

369

known as an oxidative stress inducer. After 24 h incubation, the inhibition zones 18


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370

around CHP-soaked filters were significantly smaller on the plates containing E. coli

371

cells overexpressing BoGSTd2 than in the control containing the vector only (Figure

372

6A). The halo diameter of inhibition zones was reduced more than 45% compared

373

with the control (Figure 6B).

374

ROS Level and GPOX Activity after the Exposure of Inecticides

375

The ROS level and GPOX activity of B. odoriphaga larvae after exposure to

376

different insecticides at LC30 concentrations were measured (Figure 7). Compared to

377

the control, the ROS level was increased significantly exposed to three insecticides

378

(Figure 7A). Compared to the control, GPOX activity of B. odoriphaga larvae was

379

significantly increased at 48 h post-exposure to lambda-cyhalothrin, and significantly

380

increased at 24 h, 36 h and 48 h post-exposure to chlorpyrifos and clothianidin

381

(Figure 7B).

382

RNAi of BoGSTd2

383

In order to evaluate sensitivity of dsGSTd2-treated insects to insecticides, we fed

384

B. odoriphaga third-instar larvae with dsGSTd2, and measured transcript levels of

385

BoGSTd2 at 48 h after dsRNA uptake. qRT-PCR analysis indicated that the transcript

386

level of BoGSTd2 was reduced by 64.74% (Figure 8A). We measured the

387

susceptibility of dsRNA-fed B. odoriphaga larvae to lambda-cyhalothrin, chlorpyrifos,

388

and clothianidin, respectively. There was no significant difference in mortality

389

between the dsGSTd2-treated and dsGFP-treated group to lambda-cyhalothrin at the

390

dose of LC30. Compared to dsGFP-treated larvae, the mortality of dsGSTd2-treated

391

larvae significantly increased following exposure to chlorpyrifos (from 28.9 to 41.1%) 19



392

and clothianidin (from 30.9 to 56.8%) at the LC30 dose (Figure 8B). These results

393

indicate that the silencing of BoGSTd2 gene increased the susceptibility of B.

394

odoriphaga to chlorpyrifos and clothianidin. Compared to dsGFP-treated larvae, the

395

ROS level of dsGSTd2-treated larvae was significantly increased following exposure

396

to chlorpyrifos and clothianidin at the LC30 dose. The ROS level of dsGSTd2-treated

397

larvae exposed to LC30 lambda-cyhalothrin was increased slightly, but no significant

398

change was observed compared with dsGFP-treated larvae (Figure 8C). Compared to

399

dsGFP-treated larvae, the GPOX activity of dsGSTd2-treated larvae was significantly

400

decreased following exposure to chlorpyrifos and clothianidin at the LC30 dose. The

401

GPOX activity of dsGSTd2-treated larvae exposed to LC30 lambda-cyhalothrin was

402

decreased slightly, but no significant change was observed compared with

403

dsGFP-treated larvae (Figure 8D).

404

DISCUSSION

405

Glutathione S-transferases, a family of multifunctional enzymes, is known to be

406

involved in detoxification of both xenobiotic and endogenouscompounds, including

407

insecticides and plant toxins.11,13 These enzymes are of great interest due to their role

408

in insecticide resistance. In this study, we identified and characterized one delta class

409

GST gene (BoGSTd2) from B. odoriphaga. Sequence alignment indicates that

410

BoGSTd2 shares a high identity with other known insect delta GSTs.

411

qRT-PCR was performed to assess the expression profiles of BoGSTd2. The

412

expression of BoGSTd2 was significantly higher in the fourth instar larval stage and

413

adult stage compared to other developmental stages (Figure 4A). Similar results have 20


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414

also been reported in B. dorsalis,32 in which BdGSTd6 showed higher expressions in

415

the adult and larval stages, and the relative expression levels of BdGSTd5 and

416

BdGSTd2 were significantly higher in larval stages. It is possible that in the fourth

417

instar, a relatively active feeding stage, B. odoriphaga actively intakes and digests

418

more food, and thus may be exposed to more food-borne xenobiotics. The higher

419

transcriptional levels of BoGSTd2 in the adult stage suggest that adults may suffer

420

more adverse stress from the environment, and BoGSTd2 might be expected to play

421

an important role in protection against insecticides or oxidative stress.33 The lower

422

expression of BoGSTd2 in the non-feeding pupal stage suggests that BoGSTd2 might

423

be involved in the detoxification of dietary compounds.33 Many studies have reported

424

that GST genes are expressed abundantly in insect tissues, including the midgut, fat

425

body, and Malpighian tubules where metabolic detoxification occurs.34-40 In B.

426

dorsalis, BdGSTd1 was highly expressed in the midgut and Malpighian tubules,

427

BdGSTd5 was highly expressed in the midgut, expression of BdGSTd10 was higher in

428

the Malpighian tubules, and expression of BdGSTd6 was higher in the fat body.32 Our

429

results indicated that BoGSTd2 is expressed predominantly in the midgut and

430

Malpighian tubules in fourth-instar larvae and in the abdomen of adults (Figure 4B,

431

C), where these tissues are located. These results suggest that BoGSTd2 may play

432

roles in the metabolic detoxification of xenobiotics.

433

It has been shown that a number of GSTs are involved in detoxification of

434

insecticides. Enhanced transcript level of PcGSTm5 from P. citri is correlated with

435

abamectin detoxification.41 Upregulation of BdGSTd1 and BdGSTd10 following 21



436

exposure to malathion has also been reported to be involved in malathion

437

detoxification in B. dorsalis.17 The upregulated expression of PcGSTd1 in P. citri

438

plays an antioxidant role by reducing oxidative stress caused by fenpropathrin.23 Our

439

results indicated that BoGSTd2 expression levels were significantly upregulated

440

following exposure to chlorpyrifos and clothianidin (Figure 5), suggesting that

441

BoGSTd2 might be involved in detoxification of the two insecticides in B.

442

odoriphaga.

443

Some studies have provided evidence that GSTs can metabolize or sequester

444

toxic compounds directly. BdGSTd1 or BdGSTd10 could metabolize or sequester and

445

deplete malathion directly in B. dorsalis.17 TuGSTd5 in Tetranychus urticae can

446

directly metabolize cyflumetofen.42 To investigate whether BoGSTd2 could

447

metabolize insecticides directly, recombinant BoGSTd2 was produced in E. coli BL21

448

(DE3) and further purified. The activity of BoGSTd2 was measured using CDNB as a

449

substrate. The Vmax and Km of BoGSTd2 are 349.4±10.7 μmol/min/mg and

450

0.1219±0.0123 mM, respectively, similar to those measured for AgGSTd1-6 from

451

Anopheles gambiae.43 The competitive inhibition assays showed that none of the two

452

insecticides (chlorpyrifos and clothianidin) displayed significant inhibition of

453

BoGSTd2 activity under assay conditions. This indicates that both insecticides might

454

not be able to strongly conjugate with BoGSTd2. UPLC chromatograms showed that

455

BoGSTd2 does not directly metabolize chlorpyrifos and clothianidin. The study in P.

456

citri showed that PcGSTd1 is involved in the detoxification of fenpropathrin by

457

detoxifying lipid peroxidation products caused by fenpropathrin.23 Combined with 22


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458

upregulated expression of BoGSTd2 induced by chlorpyrifos and clothianidin, we

459

speculate that BoGSTd2 may participate in the detoxification of chlorpyrifos and

460

clothianidin by other means such as protecting tissues or cells against oxidative

461

damage and oxidative stress.

462

Previous studies have shown that CpGSTd1 and CpGSTd3 in C. pomonella

463

could directly metabolize lambda-cyhalothrin.18,19 CpGSTD1 and CpGSTD2 in Culex

464

pipiens could not metabolize or sequester permethrin, however, GSTD1 was able to

465

metabolize fluorescent permethrin-like substrates.44 In our study, a delta class GST

466

gene BoGSTd2 is not responsible for lambda-cyhalothrin detoxification. Differences

467

among GSTs in detoxification function could be a result of the C-terminal domain

468

being less conserved, in which differences in the hydrophobic binding site could

469

result in modification of its insecticide binding properties. In Helicoverpa armigera,

470

although HaGST-7 and HaGST-8 exhibited minor differences of amino acid

471

sequence, the binding property of HaGST-8 with cypermethrin was substantially

472

weaker than that of HaGST-7.45

473

The ability of GSTs to metabolize lipid peroxidation products and peroxides

474

may play an important role in insect survival under oxidative stress.22 Peroxidase

475

activities of GSTs are particularly importance for insects. The recombinant BoGSTd2

476

also displayed high peroxidase activity towards CHP or H2O2, two substrates of

477

GSH-dependent peroxidase (Table 1). Disk diffusion assays indicated the inhibition

478

zones around the discs of E. coli cells overexpressing BoGSTd2 were significantly

479

smaller than the control after exposure to CHP for 24 h (Figure 8). These results 23



480

provide direct evidence that BoGSTd2 plays an important role in protection against

481

oxidative stress. Many GSTs displaying peroxidase activity have been identified that

482

may serve as antioxidant enzymes protecting organisms from oxidative damage.

483

Studies in N. lugens suggested that peroxidase activity of GSTs is positively involved

484

in pyrethroid resistance.20 The delta class (TuGSTd10, TuGSTd14, and TuGSTd05)

485

from T. urticae displayed GSH-dependent peroxidase activity toward cumene

486

hydroperoxide.9,46 AccGSTZ1 and AccGSTO2 from Apis cerana cerana and

487

RpGSTO1 from Rhopalosiphum padi may function as effective antioxidant enzymes

488

that protect cells from oxidative stress.47,48,49

489

Finally, to further confirm that BoGSTd2 play an antioxidant role in chlorpyrifos

490

and clothianidin detoxification, ROS level and GPOX activity was measured. The

491

significant increase in ROS levels following exposure to LC30 concentrations of

492

lambda-cyhalothrin, chlorpyrifos and clothianidin indicates that these three

493

insecticides elicit oxidative stress in B. odoriphaga. GPOX activity increased

494

significantly following exposure to three insecticides. This might contribute to

495

protecting B. odoriphaga from the oxidative damage caused by insecticides.

496

Compared to dsGFP-treated larvae, the ROS level and the mortality of

497

dsGSTd2-treated larvae was significantly increased, and the GPOX activity of

498

dsGSTd2-treated larvae was significantly decreased following exposure to

499

chlorpyrifos and clothianidin. However, the ROS level, the mortality and GPOX

500

activity of dsGSTd2-treated larvae exposed to lambda-cyhalothrin was not

501

significantly different compared with dsGFP-treated larvae. Combined with the direct 24


Page 24 of 46

Page 25 of 46


502

evidence from the

503

strongly indicate that BoGSTd2 participates in the elimination of ROS induced by

504

chlorpyrifos and clothianidin, but the ability of BoGSTd2 to eliminate ROS induced

505

by lambda-cyhalothrin is weak. These results suggest that change of ROS caused by

506

RNAi of dsGSTd2 may alter susceptibility of B. odoriphaga to chlorpyrifos and

507

clothianidin. Taken together, these results strongly suggest that BoGSTd2 may play an

508

important role in chlorpyrifos and clothianidin detoxification in B. odoriphaga

509

through protecting tissues from insecticide-induced oxidative stress.

disk diffusion assay, differences caused by RNAi of dsGSTd2

510 511

AUTHOR CONTRIBUTIONS

512

C.Z., B.T., and W.D. conceived and designed the experiments. B.T., L.Q., Q.Z., and

513

C.Z. performed the experiments. B.T. and C.Z. analyzed the data. C.Z. and W.D.

514

wrote the paper.

515 516

FUNDING

517

The research was supported by the National Natural Science Foundation of China

518

(31672037), the Natural Science Basic Research Plan in Shaanxi Province of China

519

(2019JM-519) and the Special Fund for Agro-scientific Research in the Public

520

Interest from the Ministry of Agriculture of China (201303027).

521 522

NOTES

523

The authors declare no competing financial interest. 25



524 525

ACKNOWLEDGEMENTS

526

We thank Dr. Chris Dietrich (University of Illinois at Urbana-Champaign, IL, USA)

527

and Dr. John Richard Schrock (Emporia State University, Emporia, KS, USA) for

528

their comments on an earlier draft of this paper.

529 530

SUPPORTING INFORMATION

531

The Supporting Information is available free of charge on the ACS Publications

532

website.

533

Table S1. Primers used in cloning of BoGSTd2, qRT-PCR and RNAi. Table S2.

534

Overview of information of the sequences in phylogenetic tree. Figure S1. cDNA and

535

deduced amino acid sequence of BoGSTd2 from B. odoriphaga. Figure S2.

536

Three-dimensional structure of BoGSTd2 from B. odoriphaga. Figure S3. Expression

537

and purification of recombinant BoGSTd2. Figure S4. Enzymatic properties of

538

recombinant BoGSTd2. Figure S5. The kinetic properties of recombinant BoGSTd2.

539

Figure S6. Dose-response curves for the inhibition of CDNB conjugating activity of

540

BoGSTd2 by insecticides. Figure S7. Chromatograms reports of insecticides

541

metabolic assays.

26


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tentans (Diptera: Chironomidae). Insect Biochem. Mol. Biol. 2009, 39(10), 745-754.

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(37) Arrese, E. L.; Soulages, J. L. Insect fat body: energy, metabolism, and regulation. Annu. Rev. Entomol. 2010, 55, 207-225. doi: 10.1146/annurev-ento-112408-085356 (38) Hakim, R. S.; Baldwin, K.; Smagghe, G. Regulation of Midgut Growth, Development, and Metamorphosis. Annu. Rev. Entomol. 2010, 55(1), 593.

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(39) Huang, Y. F.; Xu, Z. B.; Lin, X. Y.; Feng, Q. L.; Zheng, S. C. Structure and expression of

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glutathione S -transferase genes from the midgut of the Common cutworm, Spodoptera litura

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(Noctuidae) and their response to xenobiotic compounds and bacteria. J. Insect Physiol. 2011,

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57(7), 1033-1044.

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G. M.; You, M. Characterization and expression profiling of glutathione S-transferases in the

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diamondback moth, Plutella xylostella (L.). BMC Genomics 2015, 16(1), 152.

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(41) Liao, C. Y.; Xia, W. K.; Feng, Y. C.; Li, G.; Liu, H.; Dou, W.; Wang, J. J. Characterization and

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functional analysis of a novel glutathione S-transferase gene potentially associated with the

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abamectin resistance in Panonychus citri (McGregor). Pestic. Biochem. Physiol. 2016, 132, 72-80.

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G.; Labrou, N. E.; Vontas, J.; Leeuwen, T. V. A glutathione-S-transferase (TuGSTd05) associated

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with acaricide resistance in Tetranychus urticae directly metabolizes the complex II inhibitor

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cyflumetofen. Insect Biochem. Mol. Biol. 2017, 80, 101–115.

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(43) Ranson, H.; Prapanthadara, L.; Hemingway, J. Cloning and characterization of two glutathione

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S-transferases from a DDT-resistant strain of Anopheles gambiae. Biochem. J. 1997, 324(1),

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97-102.

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Manage. Sci. 2012, 68, 764– 772.

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(45) Labade, C. P.; Jadhav, A. R.; Ahire, M.; Zinjarde, S. S.; Tamhane, V. A. Role of induced

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detoxification of pesticides. Ecotoxicol. Environ. Saf. 2018, 147, 612– 621,

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Tetranychus urticae. Pestic. Biochem. Physiol. 2015, 121, 53-60.

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Under Insecticide Stress. Front. Physiol. 2018, 9, 427.

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glutathione S-transferase gene in Apis cerana cerana: molecular characterisation of GSTO2 and its

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protective effects in oxidative stress. Cell Stress Chaperon. 2013, 18(4), 503-516.

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679 680

Table 1. Kinetic parameters of recombinant BoGSTd2 Substrates

Vmax (μmol/mg/min)

Km (mM)

Kcat (s-1)

Kcat/Km (s-1 mM-1)

CDNB

349.4±10.71

0.122±0.012

160.890

1319.849

CHP

50.20±3.105

0.367±0.063

21.116

62.917

H2O2

155.90±9.329

0.415±0.064

71.788

173.150

Km, Michaelis-Menten constant; Vmax, Maximum velocity; kcat, catalytic constant; CDNB, 1-chloro-2, 4-dinitrobenzene; CHP, Cumene hydroperoxide. Values are expressed as mean ± SE.

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682 683

FIGURE LEGENDS

684

Figure 1. Alignment of amino acid sequences of GSTs from dipteran insects. Filled

685

circles indicate amino acid residues (Ser11, His52, Ala53, Val54, Glu67 and Ser68)

686

that compose the G-site and the black filled triangles indicate the amino acid residues

687

(Leu104, Tyr108, Gln109, Ile112, Asp113, Tyr116, Phe120, Thr167 and Thr170) that

688

compose the H-site in BoGSTd2 from B. odoriphaga. AdGSTd1 from Anopheles

689

darling, ETN67358.1; AgGSTd1-5 from Anopheles gambiae, CAB03592.1; CpGSTd

690

from Culex pipiens, AEW07373.1; DaGSTd1 from Delia antiqua, ALF04571.1;

691

DmGSTd1 from Drosophila melanogaster, AAB26519.1; EbGSTd1 from Episyrphus

692

balteatus, CAH58743.1; MdGSTd1 from Mayetiola destructor, ABG56084.1.

693 694

Figure 2. Phylogenetic relationships of glutathione transferases (GSTs) from different

695

insect species. The BoGSTd2 is boxed. The details and GenBank accession numbers

696

of all GSTs are shown in the Table S2.

697 698

Figure 3. Expression patterns of BoGSTd2 in different developmental stages (A),

699

various tissues of adults (B), and various tissues of fourth-instar larvae (C). The data

700

and error bars represent the means and standard error of three biological replications.

701

Different letters above the bars indicate significant differences at P< 0.05 (Student–

702

Newman–Keuls (SNK) in one way ANOVA).

703 704

Figure 4. Expression levels of BoGSTd2 under different insecticide stress. (A) 35



705

lambda-cyhalothrin; (B) chlorpyrifos; (C) clothianidin. Data and error bars represent

706

the means and standard errors of three biological replications. The expression level of

707

BoGSTd2 in the control is marked with a dash line. The “*” means significant

708

differences between the treatment and the control at the same time, * p < 0.05; ** p

Identification and Functional Analysis of a Delta Class Glutathione S

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